Practical immunodiagnostic tests for in vitro detection of analytes in fluid samples are highly specific but vary in speed, sensitivity, and the degree to which they are qualitative or quantitative. They also vary widely in assay methods used, from solution based assays to those that are formulated on solid supports, such as beads, membranes, lateral flow strips, microtiter plates, biochips, and planar or semi-planar substrates such as coated slides. Quantitative immunoassays are usually associated with automated platforms designed to run high-throughput assays in a clinical environment. These systems are typically designed to measure a single analyte for each sample-test. Other assay methods such as ELISA plate and lateral flow assay can also be automated but are limited in the ability to multiplex.
Common methods used for inexpensive, qualitative testing are the dipstick and lateral flow strip formats. These assays are also typically designed to test for a single, or at most only a few, analytes per assay. Such test devices are usually designed to produce a visual signal on the solid support that can be observed by the naked eye, providing only a qualitative, or at best a semi-quantitative, test result. The sensitivity of the lateral flow format is generally maximized by increasing the strength of the visible signal, which decreases the lower limit of detection of the assay (LLD). In some cases, however, optimizing the visual signal creates an assay that is too sensitive, because the assays detect a concentration of analyte below the desired detection level, thereby leading to false positives. The sensitivity of the assay no-longer matches the concentration range over which one wishes to detect the analyte. In such cases, lateral flow assays have been adjusted by adding small increments of adjustment antibodies to offset the signal, so that a positive test signal is only observed when the analyte is present at or above a predetermined threshold.
A new generation of immunoassays is being developed to better meet the needs of clinicians and researchers. Multiplexed immunoassays are designed to measure multiple (e.g., as high as 42 or more) analytes in a single-sample test. These assays provide the potential to reduce costs and minimize sample volumes in certain volume-sensitive applications. Sample volume requirement is reduced by measuring a number of analytes in a single sample. Examples of sample-limited applications include pediatric testing in diagnostics, animal model testing in research, and the screening of serum banks to develop disease-specific biomarkers. In each of these cases, the volume of sample available for testing is limited. Microarray technology provides a promising platform for assaying multiplexed immunoassays that can potentially provide more sensitive and quantitative methods for measuring the concentration of multiple analytes within a single assay. The term “microarray” refers to a solid, planar or semi-planar substrate on which are arranged a set of microscopic spatially distinct areas, or spots, to which are attached one or more capture molecules. The intensity of the signal resulting from the assay is quantified using an optical imager.
Two parameters that are often used to describe the capability of a microarray assay are the lowest level of detection (LLD) and the working range. The LLD is the lowest analyte concentration that can be reliably quantified in the assay, and is a measure of the sensitivity of the assay. The working range, also variously referred to as the dynamic range or the assay range, is the range of analyte concentrations over which a change in concentration can be reliably measured, usually the range over which the optical signal is a linear function of analyte concentration. By way of example, an assay for the cytokine IL-8 having an LLD of 10 pg/mL and a working range of three logs provides a reliable quantitative measure of concentrations between 10 pg/mL and 10 ng/mL, but would not be acceptable to a researcher who is interested in IL-8 over a range of 500 pg/mL to 50 ng/mL. The assay would have adequate sensitivity at the low end of the working range (10 pg/mL), but would not discriminate IL-8 concentrations above 10 ng/mL and therefore would not meet the high-end requirements.
When a single analyte assay is too sensitive, the sample can be diluted to adjust the expected analyte range to the assay range. Dilution techniques are not suitable, however, in the case of a multiplexed immunoassay, which attempts independent, simultaneous measurement of multiple analytes in a single sample. By way of example, consider a simple two-plex immunoassay (two antigenic proteins detected in a single sample) having a working range of 10 pg/mL to 10 ng/mL for both analytes. Consider the application of this assay for detecting IL-7 over the range of 20 pg/mL to 2 ng/mL, and detecting IL-8 over the range of 500 pg/mL to 50 ng/mL. The working range of the assay is well suited to detect the target range of concentration for IL-7, but the targeted IL-8 concentration exceeds the upper end of the working range. Were the sample to be diluted (e.g., by 1:5), the IL-8 would be within the working range, but the IL-7 concentration would be below the low end of the assay working range (FIG. 1). The situation is further exacerbated when the number of analytes tested is increased to 10, 20 or higher. Thus, simple dilution methods do not have enough degrees of freedom to adjust the working range of the assay independently for each analyte in a multiplexed assay. What is needed is a method to independently match the range of each analyte to the assay range in a multiplexed immunoassay, preferably over several orders of magnitude, preferably over a range of three, four, five or six orders of magnitude.